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Down-Regulation of the INK4 Family of Cyclin-Dependent Kinase Inhibitors by Tax Protein of HTLV-1 Through Two Distinct Mechanisms

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Down-Regulation of the INK4 Family of Cyclin-Dependent Kinase Inhibitors by Tax Protein of HTLV-1 Through Two Distinct Mechanisms Virology 259, 384–391 (1999) Article ID viro.1999.9760, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Down-regulation of the INK4 Family of Cyclin-Dependent Kinase Inhibitors by Tax Protein of HTLV-1 through Two Distinct Mechanisms Takeshi Suzuki, Tomoko Narita, Masami Uchida-Toita, and Mitsuaki Yoshida1 Department of Cellular and Molecular Biology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received January 18, 1999; returned to author for revision March 8, 1999; accepted April 9, 1999 Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) affects multiple regulatory processes of infected cells through activation and repression of specific transcription and also through modulation of functions of cell cycle regulators. Previously, we found that Tax binds to p16ink4a, a member of the INK4 family of cyclin-dependent kinase inhibitors, and counteracts its inhibitory activity, resulting in cell cycle progression. In this study, we examined the effects of Tax on other members of the INK4 family and found that Tax can bind to p15ink4b similarly to p16ink4a, but not to p18ink4c and p19ink4d. Tax binding to p15ink4b inactivated its function and restored CDK4 kinase activity. Accordingly, Tax-expressing cells became resistant to p15ink4b-mediated growth arrest induced by TGFb. On the other hand, expression of p18ink4c was transcrip- tionally repressed by Tax through the E-box element of the promoter, which may contribute to the marked reduction of p18ink4c mRNA in HTLV-1-infected T-cells. These observations indicate that Tax suppresses the inhibitory activities of INK4 family members through two independent mechanisms: functional inhibition of two INK4 proteins and repression of expression of another INK4 protein. These effects may play roles in HTLV-1-induced deregulation of the cell cycle, possibly promoting cellular transformation. © 1999 Academic Press INTRODUCTION Reddy, 1995; Hunter and Pines, 1994). Tax protein of HTLV-1 binds to one of the INK4 family members, Human T-cell leukemia virus type 1 (HTLV-1) (Poeisz et p16ink4a, and suppresses its inhibitory activity, causing al., 1980; Yoshida et al., 1982) is the causative agent of a activation of CDK4. Tax consequently rescued cells from unique T-cell malignancy, adult T-cell leukemia (ATL) G1 arrest, which was induced by overexpression of (Hinuma et al., 1981; Uchiyama et al., 1977). The virally p16ink4a, and resulted in cell proliferation (Suzuki et al., encoded Tax protein is required for viral replication and 1996). Thus, these results provided a novel insight into has been suggested to contribute to the oncogenesis the abnormal regulation induced by HTLV-1 infection. associated with HTLV-1 infection (Yoshida, 1993). Tax The INK4 family consists of p16ink4a (Serrano et al., was originally identified as a transcriptional activator on 1993), p15ink4b (Hannon and Beach, 1994), p18ink4c, the one hand and as a repressor on the other hand, and p19ink4d (Chan et al., 1995; Guan et al., 1994; Hirai affecting specific cellular gene expression (Brauweiler et al., 1997; Inoue et al., 1986; Jeang et al., 1990; Lemasson et al., 1995), all of which have ankyrin motifs. Since Tax et al., 1997; Yoshida, 1995). In addition to transcriptional can bind to the ankyrin motifs of p16ink4a, we examined regulation, Tax was found to inactivate a cyclin-depen- the effects of Tax on INK4-mediated regulation. In this dent kinase (CDK) inhibitor, p16ink4a protein, and dereg- paper, we describe two independent mechanisms by ulate cell cycle control (Suzuki et al., 1996). which Tax inhibited the regulation controlled by the INK4 Cell cycle progression in eukaryotes is controlled by family: direct binding to p15ink4b and transcriptional sequential activation and subsequent inactivation of a repression of p18ink4c expression. Tax binding to series of CDKs at specific points of the cell cycle (Pines, p15ink4b inactivated its function and made cells resis- 1993; Sherr, 1994). The activities of CDKs are positively tant to p15ink4b-mediated growth arrest induced by b regulated by cyclins and negatively regulated by a family TGF . On the other hand, Tax did not bind to p18ink4c of CDK inhibitors including the INK4 family (Grana and and p19ink4d, but repressed transcription of p18ink4c gene through the E-box element of the promoter. Identi- fication of the E-box as a responsible element for tran- 1 scriptional repression is consistent with recent reports To whom correspondence and reprint requests should be ad- b dressed at present address: Banyu Tsukuba Research Institute, Okubo on Tax-mediated trans-repression of DNA polymerase 3, Tsukuba, Ibaraki 300-2611, Japan. Fax: 81-298-77-2034. E-mail: and the lck and bax genes (Brauweiler et al., 1997; [email protected]. Lemasson et al., 1997; Uittenbogaard et al., 1994). The 0042-6822/99 $30.00 Copyright © 1999 by Academic Press 384 All rights of reproduction in any form reserved. EFFECTS OF TAX ON INK4 FAMILY PROTEINS 385 suppression of the function and expression of INK4 fam- ily members possibly contributes to cell cycle deregula- tion in HTLV-1-infected cells. RESULTS Interaction of Tax with INK4 family proteins Previously, we have demonstrated binding of Tax to p16ink4a protein (Suzuki et al., 1996), a member of the INK4 family of CDK inhibitors which contains four tandem repeats of the ankyrin motif (Serrano et al., 1993). Other members of the INK4 family, p15ink4b, p18ink4c, and p19ink4d, all contain similar ankyrin motifs to p16ink4a FIG. 2. Effect of p15ink4b and Tax on CDK4 kinase activity. Expres- and inhibit CDK4 activity (Grana and Reddy, 1995). Thus sion vectors for cyclin D1 (lanes 1–4), CDK4 (lanes 1–4), p15ink4b we were interested in whether Tax interacts with these (lanes 2–4), wild-type Tax (lane 3), and the d7/16 mutant (lane 4) were members of the INK4 family. We produced fusion pro- cotransfected into 293T cells. Total cell extracts were treated with anti-CDK4 antibody, and the immunoprecipitates were subjected to teins of INK4 family members with glutathione–S-trans- kinase assay by CDK4 using GST-pRb as the substrate followed by ferase (GST) and analyzed their binding to recombinant SDS–PAGE and autoradiography. Tax protein (His-Tax). The GST fusion proteins were ad- sorbed on glutathione–Sepharose beads and mixed with purified His-Tax. The complexes were collected by cen- action on CDK4 activity (Suzuki et al., 1996). To examine trifugation and subjected to Western blotting for detec- the similar effect of Tax on p15ink4b, the kinase activity tion of Tax protein. As shown in Fig. 1, Tax was detected of CDK4 was assayed in vitro using GST-pRb as a sub- in the complexes with GST-p15ink4b as well as GST- strate. Expression vectors for cyclin D1, CDK4, p15ink4b, p16ink4a (lanes 3 and 5), but not with GST alone (lane 1), and Tax were cotransfected into 293T cells and the cell GST-p18ink4c, or GST-p19ink4d (lanes 7 and 9). Tax mu- extracts were treated with anti-CDK4 antibody. The im- tant d7/16, which did not bind to p16ink4a, was not mune complexes were subjected to in vitro kinase as- detected in the complexes with p15ink4b, confirming says by adding GST-pRb and [g32P]ATP, and the phos- binding specificity in vitro. phorylation of GST-pRb was analyzed by SDS–gel elec- trophoresis (Matsushime et al., 1994). When cyclin D1 Cancellation of p15ink4b-mediated inhibition of CDK4 and CDK4 were transfected, significant phosphorylation by Tax of GST-pRb was detected (Fig. 2, lane 1), but cotransfec- INK4 family proteins bind to cyclin D-dependent cata- tion of p15ink4b resulted in a decreased phosphorylation lytic subunit CDK4 or CDK6 and inhibit their kinase ac- (lane 2), consistent with the inhibitory effect of p15ink4b tivity (Grana and Reddy, 1995; Hunter and Pines, 1994). on CDK4 activity. Additional expression of Tax in this The binding of Tax to p16ink4a counteracts its inhibitory system restored the CDK4 kinase activity (lane 3), which had been suppressed by the expression of p15ink4b. These results indicate that Tax counteracted the inhibi- tory effect of p15ink4b on CDK4 kinase activity, similarly to p16ink4a. The inactive Tax mutant d7/16, which could not bind to p15ink4b, did not show any effect (lane 4), suggesting that the effect was mediated through the binding of Tax to p15ink4b. Inhibition of TGFb-induced arrest of cell growth p15ink4b was originally identified as a CDK inhibitor induced by TGFb (Hannon and Beach, 1994), which ar- FIG. 1. Binding of Tax protein to INK4 family proteins in vitro. Five rests the cell cycle at G1 (Reynisdottir et al., 1995). To nanograms of purified His-Tax (lanes 1, 3, 5, 7, 9) or its mutant d7/16 demonstrate the effect of Tax on p15ink4b-induced (lanes 2, 4, 6, 8, 10) was mixed with glutathione–Sepharose beads growth arrest, the expression vector for Tax was trans- m containing 2 g each of GST (lanes 1, 2), GST-p15ink4b (lanes 3, 4), fected with a puromycin resistance gene into mouse GST-p16ink4a (lanes 5, 6), GST-p18ink4c (lanes 7, 8), or GST-p19ink4d (lanes 9, 10) and incubated for8hat4°C. The bound Tax was collected BaF3 cell line, and cell clones expressing Tax protein by centrifugation and analyzed by Western blotting with anti-Tax anti- were isolated. Tax-expressing BaF3 cells and control body. BaF3 cells were treated with increasing concentrations 386 SUZUKI ET AL. of TGFb for 10 h. The proliferation of cells was monitored by incorporation of BrdU into newly synthesized DNA. Treatment of control cells with TGFb decreased BrdU incorporation, indicating that TGFb induced cell growth arrest (Fig. 3A).
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